Novel maytansinoid derivatives with sulfoxide linker

ABSTRACT

The invention relates to novel maytansinoid compounds having sulfoxide linkers and more specifically to novel maytansinoid compounds of structural formula (I) and (II). The invention also provides conjugates of the maytansinoid compounds linked to a cell-binding agent. The invention further provides compositions and methods useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal using the compounds or conjugates of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/009/957, filed Oct. 4, 2013 (now allowed); which is a National Stageof International Application No. PCT/US2012/029791 filed Mar. 20, 2012,claiming priority based on U.S. Provisional Application No. 61/504,953filed Jul. 6, 2011, and U.S. Provisional Application No. 61/476,529filed Apr. 18, 2011; the contents of all of which are incorporatedherein by reference in their entirety

FIELD OF THE INVENTION

The present invention relates to novel cytotoxic compounds and cytotoxicconjugates comprising these cytotoxic compounds and cell-binding agents.More specifically, this invention relates to novel maytansinoidcompounds, derivatives thereof, intermediates thereof, conjugatesthereof, and pharmaceutically acceptable salts thereof, which are usefulas medicaments, in particular as anti-proliferative agents.

BACKGROUND OF THE INVENTION

Antibody-drug conjugates (ADC) are emerging as a powerful class ofanti-tumor agents with efficacy across a range of cancers. ADCs arecommonly composed of three distinct elements: a cell-binding agent; alinker; and a cytotoxic agent. The linker component of ADC is animportant element in developing targeted anti-cancer agents that possessan optimal therapeutic window: high activity at a low, non-toxic dose.

Therefore, there is a need for ADCs having new class of linkercomponent.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed to a compoundrepresented by structural formula (I′):

wherein:

-   -   D is a cytotoxic agent;    -   X is S or Se;    -   R and R′ are each independently selected from the group        consisting of —H, an alkyl, a cycloalkyl, —OR^(a), and        —NR^(b)R^(c);    -   R^(a), R^(b) and R^(c) are each independently H or an alkyl;    -   p is 0, 1 or 2    -   Y″ is a spacer;    -   M is a linking group that can react with a cell-binding agent to        form a covalent bond.

One embodiment of the present invention is directed to a compoundrepresented by structural formula (I):

wherein:

-   -   D is a cytotoxic agent;    -   X is S or Se;    -   R and R′ are each independently selected from the group        consisting of —H, an alkyl, a cycloalkyl, —OR^(a), and        —NR^(b)R^(c);    -   R^(a), R^(b) and R^(c) are each independently H or an alkyl;    -   p is 0, 1 or 2    -   Y is an alkylene, a polyethylene glycol unit represented by        —(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or —R^(d)—W—R^(e)—,    -   n is an integer from 1 to 24;    -   E is a cycloalkyl, a heterocyclyl, an aryl or a heteroaryl,    -   W is —C(═O)NH—, —NHC(═O)—, —(C═O)O— or —O(C═O)—,    -   R^(d) is absent or an alkyl;    -   R^(e) is an alkyl;    -   M is a linking group that can react with a cell-binding agent to        form a covalent bond.

In another embodiment, the present invention is directed to a compoundof structural formula (II):

wherein:

-   -   D is a cytotoxic agent;    -   X is S or Se;    -   Y′ is —SO₂—, aryl, pyridyl or —C(═O)—;    -   Z is an alkyl, a cycloalkyl, a heterocyclyl, an aryl, a        heteroaryl, or —N(R^(b)′)—R^(d)′—;    -   R^(b)′ is H or a C₁₋₄ alkyl;    -   R^(d)′ is a C₁₋₄ alkyl; and    -   M is a linking group that can react with a cell-binding agent to        form a covalent bond.

The present invention is also directed to conjugates comprising acell-binding agent chemically linked to a compound of structural formula(I′), (I) or (II). In one embodiment, the conjugate is represented bythe following structural formula:

wherein:

-   -   CBA represents a cell-binding agent;    -   D is a cytotoxic agent;    -   X is S or Se;    -   R and R′ are each independently selected from the group        consisting of —H, an alkyl, a cycloalkyl, —OR^(a), and        —NR^(b)R^(c);    -   R^(a), R^(b) and R^(c) are each independently H or an alkyl;    -   p is 0, 1 or 2;    -   Y″ is a spacer;    -   q is an integer from 1 to 20; and    -   M′ is a linking moiety.

In another embodiment, the conjugate is represented by the followingstructural formula:

wherein:

-   -   CBA represents a cell-binding agent;    -   D is a cytotoxic agent;    -   X is S or Se;    -   R and R′ are each independently selected from the group        consisting of —H, an alkyl, a cycloalkyl, —OR^(a), and        —NR^(b)R^(c);    -   R^(a), R^(b) and R^(c) are each independently H or an alkyl;    -   p is 0, 1 or 2    -   Y is an alkylene, a polyethylene glycol unit represented by        —(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or —R^(d)—W—R^(e)—,    -   n is an integer from 1 to 24;    -   E is a cycloalkyl, a heterocyclyl, an aryl or a heteroaryl,    -   W is —C(═O)NH—, —NHC(═O)—, —(C═O)O— or —O(C═O)—,    -   R^(d) is absent or an alkyl;    -   R^(e) is an alkyl;    -   q is an integer from 1 to 20; and    -   M′ is a linking moiety.

In another embodiment, the conjugate is represented by the followingstructural formula:

wherein:

-   -   D is a cytotoxic agent;    -   X is S or Se;    -   Y′ is —SO₂—, aryl, pyridyl or —C(═O)—;    -   Z is an alkyl, a cycloalkyl, a heterocyclyl, an aryl, a        heteroaryl, or —N(R^(b)′)—R^(d)′—;    -   R^(b)′ is H or a C₁₋₄ alkyl;    -   R^(d)′ is a C₁₋₄ alkyl;    -   q is an integer from 1 to 20; and    -   M′ is a linking moiety.

The present invention is also directed to a pharmaceutical compositioncomprising the conjugates described herein and a pharmaceuticallyacceptable carrier.

In another embodiment, the present invention is directed to a method ofinhibiting abnormal cell growth or treating a proliferative disorder, anautoimmune disorder, a destructive bone disorder, an infectious disease,a viral disease, a fibrotic disease, a neurodegenerative disorder, apancreatitis or kidney disease in a mammal comprising administering tothe mammal a therapeutically effective amount of the conjugate describedherein and optionally in combination with another chemotherapeuticagent.

In another embodiment, the present invention is directed to a cytotoxiccompound or a conjugate described herein for use in inhibiting abnormalcell growth or in treating a proliferative disorder, an autoimmunedisorder, a destructive bone disorder, an infectious disease, a viraldisease, a fibrotic disease, a neurodegenerative disorder, apancreatitis or kidney disease in a mammal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows in vitro cytotoxicity of IgG1-SMCC-sulfoxide-DM1 conjugate(2a) having a sulfoxide linker as compared to IgG1-SMCC-DM1 conjugate(1a) having a thioether linker in squamous cell carcinoma cell lineA431.

FIG. 2 shows in vitro cytotoxicity of anti-EpCAM-SMCC-sulfoxide-DM1conjugate (2b) having a sulfoxide linker as compared toanti-EpCAM-SMCC-DM1 conjugate (1b) having a thioether linker in lungcarcinoma cell line PC9.

FIG. 3 depicts a hypothetical scheme for explaining the observed higherantigen-specific activity of conjugate (2) as compared to conjugate (1).

FIG. 4 shows HPLC and MS data indicating the formation of DM1-dimedone(DM1-NME) upon free maytansinoid release from the oxidized Ab-SMCC-DM1conjugate 2 after 12 h at 37° C.

FIGS. 5A-5D show rate comparison of model thioether oxidation (A), modelsulfoxide elimination (B) and free maytansinoid formation forAb-PEG₄-mal-DM_(x) conjugate (C) for unhindered DM1 and hindered DM4maytansinoid thioethers. (D) shows a reaction scheme for oxidationpromoted maytansinoid release from Ab-PEG₄-mal-DM_(x).

FIGS. 6A-6C show HPLC traces (A), ESI-MS data (B), and rate ofconversion (C) of DM1 SO-NEM to DM1-SO3- and DM1-S-SO-DM1 under elevatedtemperature (37 C) and/or reducing conditions (dithiothreitol) in PBS pH7.4. Control compound DM1-NEM was stable under these tested reactionconditions.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention.

Definitions

“Alkyl” as used herein refers to a saturated aliphatic linear orbranched-chain monovalent hydrocarbon radical having one to twentycarbon atoms. Examples of alkyl include, but are not limited to, methyl,ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, —CH₂CH(CH₃)₂,2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl 3-pentyl,2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and thelike. Preferably, the alkyl has one to ten carbon atoms, also referredto as “C₁₋₁₀ alkyl”. More preferably, the alkyl has one to six carbonatoms, also referred to as “C₁₋₆ alkyl”. Even more preferably, the alkylhas one to four carbon atoms, also referred to as “C₁₋₄ alkyl”.

“Alkenyl” as used herein refers to aliphatic linear or branched-chainmonovalent hydrocarbon radical of two to twenty carbon atoms with atleast one site of unsaturation, i.e., a carbon-carbon, double bond,wherein the alkenyl radical includes radicals having “cis” and “trans”orientations, or alternatively, “E” and “Z” orientations. Examplesinclude, but are not limited to, ethylenyl or vinyl (—CH═CH₂), allyl(—CH₂CH═CH₂), and the like. Preferably, the alkenyl has two to tencarbon atoms, also referred to as “C₂₋₁₀ alkenyl”. More preferably, thealkyl has two to four carbon atoms, also referred to as “C₂₋₄ alkenyl”.

“Alkynyl” as used herein refers to aliphatic linear or branched-chainmonovalent hydrocarbon radical of two to twenty carbon atoms with atleast one carbon-carbon triple bond. Examples include, but are notlimited to ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,2-pentynyl, 3-pentynyl, hexynyl, and the like. Preferably, the alkynylhas two to ten carbon atoms, also referred to as “C₂₋₁₀ alkynyl”. Morepreferably, the alkynyl has two to four carbon atoms, also referred toas “C₂₋₄ alkynyl”.

The term “alkylene” refers to a saturated aliphatic linear orbranched-chain divalent hydrocarbon radical having one to twenty carbonatoms. Preferably, the alkylene has one to ten carbon atoms. Morepreferably, the alkylene has one to four carbon atoms.

The term “carbocycle”, “carbocyclyl” and “carbocyclic ring” refer to amonovalent non-aromatic, saturated or partially unsaturated ring having3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as abicyclic ring. Bicyclic carbocycles having 7 to 12 atoms can bearranged, for example, as a bicyclo[4,5], [5,5], [5,6] or [6,6] system,and bicyclic carbocycles having 9 or 10 ring atoms can be arranged as abicyclo[5,6] or [6,6] system, or as bridged systems such asbicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane.Examples of monocyclic carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-I-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-I-enyl,1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and thelike.

The terms “cyclic alkyl” and “cycloalkyl” can be used interchangeably.They refer to a monovalent saturated carbocyclic ring radical.Preferably, the cycloalkyl is 3 to 7 membered monocyclic ring radical.More preferably, the cycloalkyl is cyclohexyl.

“Aryl” as used herein means a monovalent monocyclic or polycyclic (e.g.bicyclic or tricyclic) aromatic hydrocarbon radical of 6-18 carbon atomsderived by the removal of one hydrogen atom from a single carbon atom ofa parent aromatic ring system. Some aryl groups are represented in theexemplary structures as “Ar”. Aryl includes bicyclic radicals comprisingan aromatic ring fused to a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Typical aryl groups include,but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, indenyl, indanyl,1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the like.Preferably, aryl is phenyl group.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 18 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus, and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles aredescribed in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, and azabicyclo[2.2.2]hexanyl. Spiromoieties are also included within the scope of this definition. Examplesof a heterocyclic group wherein ring atoms are substituted with oxo (═O)moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl.

The term “heteroaryl” refers to a monovalent aromatic radical of 5- or6-membered rings, and includes fused ring systems (at least one of whichis aromatic) of 5-18 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl.

The heterocycle or heteroaryl groups may be carbon (carbon-linked) ornitrogen (nitrogen-linked) attached where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or O-carboline.

The heteroatoms present in heteroaryl or heterocyclcyl can include theoxidized forms such as NO, SO, and SO₂.

The term “halo” or “halogen” refers to F, Cl, Br or I.

The alkyl, alkenyl, cycloalkyl, carbocyclyl, aryl, heterocyclyl andheteroaryl described above can be optionally substituted with one more(e.g., 2, 3, 4, 5, 6 or more) suitable substituents. In one embodiment,the alkyl, alkenyl, cycloalkyl, carbocyclyl, aryl, heterocyclyl andheteroaryl described above is unsubstituted. In another embodiment, thealkyl, alkenyl, cycloalkyl, carbocyclyl, aryl, heterocyclyl andheteroaryl described above are substituted with one more (e.g., 2, 3, 4,5, 6 or more) suitable substituents.

Such suitable substituents, in non-limiting examples, can be selectedfrom an alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms,cycloalkyl, aryl, heteroaryl, heterocycyclyl, halogen, guanidinium[—NH(C═NH)NH₂], —OR¹⁰⁰, NR¹⁰¹R¹⁰², —NO₂, —NR¹⁰¹COR¹⁰², —SR¹⁰⁰, asulfoxide represented by —SOR¹⁰¹, a sulfone represented by —SO₂R¹⁰¹, asulfonate —SO₃M, a sulfate —OSO₃M, a sulfonamide represented by—SO₂NR¹⁰¹R¹⁰², cyano, an azido, —COR¹⁰¹, —OCOR¹⁰¹, —OCONR¹⁰¹R¹⁰² and apolyethylene glycol unit (—OCH₂CH₂)_(n)R¹⁰¹ wherein M is H or a cation(such as Na⁺ or K⁺); R¹⁰⁰, R¹⁰¹, R¹⁰² and R¹⁰³ are each independentlyselected from H, alkyl, alkenyl or alkynyl having from 1 to 10 carbonatoms, a polyethylene glycol unit (—OCH₂CH₂)_(n)—R¹⁰⁴, wherein n is aninteger from 1 to 24, an aryl having from 6 to 10 carbon atoms, aheterocyclic ring having from 3 to 10 carbon atoms and a heteroarylhaving 5 to 10 carbon atoms; and R¹⁰⁴ is H or a linear or branched alkylhaving 1 to 4 carbon atoms, wherein the alkyl, alkenyl, alkynyl, aryl,heteroaryl and heterocycyclyl in the groups represented by R¹⁰⁰, R¹⁰¹,R¹⁰², R¹⁰³ and R¹⁰⁴ are optionally substituted with one or more (e.g.,2, 3, 4, 5, 6 or more) substituents independently selected from halogen,—OH, CN, NO₂ and unsubstituted linear or branched alkyl having 1 to 4carbon atoms. Preferably, the substituents for the optionallysubstituted alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl,cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl describedabove include halogen, CN, NR¹⁰²R¹⁰³, CF₃, OR¹⁰¹, aryl, heteroaryl,heterocycycl, SR¹⁰¹, SOR¹⁰¹, SO₂R¹⁰¹ and —SO₃M. Preferably, the suitablesubstituent is selected from the group consisting of -halogen, —OH,—NO₂, —CN, C₁₋₄ alkyl, —OR¹⁰⁰, NR¹⁰¹R¹⁰², —NR¹⁰¹COR¹⁰², —SR¹⁰⁰,—SO₂R¹⁰¹, —SO₂NR¹⁰¹R¹⁰², —COR¹⁰¹, —OCOR¹⁰¹ and —OCONR¹⁰¹R¹⁰² whereinR¹⁰⁰, R¹⁰¹ and R¹⁰² are each independently —H or C₁₋₄ alkyl.

The term “compound” and “cytotoxic compound” are used interchangeably.They are intended to include compounds for which a structure or formulaor any derivative thereof has been disclosed in the present invention ora structure or formula or any derivative thereof that has beenincorporated by reference. The term also includes, stereoisomers,geometric isomers, tautomers, solvates, metabolites, salts (e.g.,pharmaceutically acceptable salts) and prodrugs, and prodrug salts of acompound of all the formulae disclosed in the present invention. Theterm also includes any solvates, hydrates, and polymorphs of any of theforegoing. The specific recitation of “stereoisomers”, “geometricisomers”, “tautomers”, “solvates”, “metabolites”, “salt” “prodrug,”“prodrug salt,” “conjugates,” “conjugates salt,” “solvate,” “hydrate,”or “polymorph” in certain aspects of the invention described in thisapplication shall not be interpreted as an intended omission of theseforms in other aspects of the invention where the term “compound” isused without recitation of these other forms.

The term “conjugate” as used herein refers to a compound describedherein or a derivative thereof that is linked to a cell binding agent.

The term “linkable to a cell binding agent” as used herein refers to thecompounds described herein or derivates thereof comprising at least onelinking group or a precursor thereof suitable to bond these compounds orderivatives thereof to a cell binding agent.

The term “precursor” of a given group refers to any group which may leadto that group by any deprotection, a chemical modification, or acoupling reaction.

The term “linked to a cell binding agent” refers to a conjugate moleculecomprising at least one of the compounds described herein (e.g.,compounds of formula (I) or (II)),or derivative thereof bound to a cellbinding agent via a suitable linking group or a precursor thereof.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their minorimage partner.

The term “stereoisomer” refers to compounds which have identicalchemical constitution and connectivity, but different orientations oftheir atoms in space that cannot be interconverted by rotation aboutsingle bonds.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not minor images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as crystallization, electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable minor images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand I or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or l meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are minor images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “prodrug” as used in this application refers to a precursor orderivative form of a compound of the invention that is capable of beingenzymatically or hydrolytically activated or converted into the moreactive parent form. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy”Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast(1986) and Stella et al., “Prodrugs: A Chemical Approach to TargetedDrug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press (1985). The prodrugs of this invention include,but are not limited to, ester-containing prodrugs, phosphate-containingprodrugs, thiophosphate-containing prodrugs, sulfate-containingprodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, .beta.-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs, optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, compounds of the invention and chemotherapeutic agents suchas described above.

The term “prodrug” is also meant to include a derivative of a compoundthat can hydrolyze, oxidize, or otherwise react under biologicalconditions (in vitro or in vivo) to provide a compound of thisinvention. Prodrugs may only become active upon such reaction underbiological conditions, or they may have activity in their unreactedforms. Examples of prodrugs contemplated in this invention include, butare not limited to, analogs or derivatives of compounds of any one ofthe formulae disclosed herein that comprise biohydrolyzable moietiessuch as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzablecarbamates, biohydrolyzable carbonates, biohydrolyzable ureides, andbiohydrolyzable phosphate analogues. Other examples of prodrugs includederivatives of compounds of any one of the formulae disclosed hereinthat comprise —NO, —NO₂, —ONO, or —ONO₂ moieties. Prodrugs can typicallybe prepared using well-known methods, such as those described byBurger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982(Manfred E. Wolff ed., 5th ed); see also Goodman and Gilman's, ThePharmacological basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed.1992, “Biotransformation of Drugs”.

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide”, “biohydrolyzable ester”, “biohydrolyzablecarbamate”, “biohydrolyzable carbonate”, “biohydrolyzable ureide” and“biohydrolyzable phosphate analogue” mean an amide, ester, carbamate,carbonate, ureide, or phosphate analogue, respectively, that either: 1)does not destroy the biological activity of the compound and confersupon that compound advantageous properties in vivo, such as uptake,duration of action, or onset of action; or 2) is itself biologicallyinactive but is converted in vivo to a biologically active compound.Examples of biohydrolyzable amides include, but are not limited to,lower alkyl amides, .alpha.-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable estersinclude, but are not limited to, lower alkyl esters, alkoxyacyloxyesters, alkyl acylamino alkyl esters, and choline esters. Examples ofbiohydrolyzable carbamates include, but are not limited to, loweralkylamines, substituted ethylenediamines, amino acids,hydroxyalkylamines, heterocyclic and heteroaromatic amines, andpolyether amines. Particularly favored prodrugs and prodrug salts arethose that increase the bioavailability of the compounds of thisinvention when such compounds are administered to a mammal.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt mayinvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion may be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

As used herein, the term “solvate” means a compound which furtherincludes a stoichiometric or non-stoichiometric amount of solvent suchas water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate,acetic acid, and ethanolamine dichloromethane, 2-propanol, or the like,bound by non-covalent intermolecular forces. Solvates or hydrates of thecompounds are readily prepared by addition of at least one molarequivalent of a hydroxylic solvent such as methanol, ethanol,1-propanol, 2-propanol or water to the compound to result in solvationor hydration of the imine moiety.

The term “cytotoxic agent” as used herein refers to any compound thatresults in the death of a cell, induces cell death, or decreases cellviability. Suitable cytotoxic agents include, for example, maytansinoidsand maytansinoid analogs, taxoids, CC-1065 and CC-1065 analogs, anddolastatin and dolastatin analogs. In a preferred embodiment of theinvention, the cytotoxic agent is a maytansinoid, including maytansinol,maytansinol analogs, ansamitocin and ansamitocin analogs. Maytansinoidsare compounds that inhibit microtubule formation and are highly toxic tomammalian cells. Examples of suitable maytansinol analogues includethose having a modified aromatic ring and those having modifications atother positions. Such maytansinoids are described in, for example, U.S.Pat. Nos. 4,256,746, 4,294,757, 4,307,016, 4,313,946, 4,315,929,4,322,348, 4,331,598, 4,361,650, 4,362,663, 4,364,866, 4,424,219,4,371,533, 4,450,254, 5,475,092, 5,585,499, 5,846,545, and 6,333,410.

Examples of maytansinol analogs having a modified aromatic ring include:(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reductionof ansamytocin P2), (2) C-20-hydroxy (or C-20-demethyl) +/−C-19-dechloro(U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylationusing Streptomyces or Actinomyces or dechlorination using LAH), and (3)C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Examples of maytansinol analogs having modifications of positions otherthan an aromatic ring include: (1) C-9-SH (U.S. Pat. No. 4,424,219)(prepared by the reaction of maytansinol with H₂S or P₂S₅), (2)C-14-alkoxymethyl (demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598), (3)C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia), (4) C-15-hydroxy/acyloxy (U.S. Pat.No. 4,364,866) (prepared by the conversion of maytansinol byStreptomyces), (5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929)(isolated from Trewia nudiflora), (6) C-18-N-demethyl (U.S. Pat. Nos.4,362,663 and 4,322,348) (prepared by the demethylation of maytansinolby Streptomyces), and (7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (preparedby the titanium trichloride/LAH reduction of maytansinol).

The terms “abnormal cell growth” and “proliferative disorder” are usedinterchangeably in this application. “Abnormal cell growth”, as usedherein, unless otherwise indicated, refers to cell growth that isindependent of normal regulatory mechanisms (e.g., loss of contactinhibition). This includes, for example, the abnormal growth of: (1)tumor cells (tumors) that proliferate by expressing a mutated tyrosinekinase or overexpression of a receptor tyrosine kinase; (2) benign andmalignant cells of other proliferative diseases in which aberranttyrosine kinase activation occurs; (3) any tumors that proliferate byreceptor tyrosine kinases; (4) any tumors that proliferate by aberrantserine/threonine kinase activation; and (5) benign and malignant cellsof other proliferative diseases in which aberrant serine/threoninekinase activation occurs.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, acute leukemia, as well as head/brain andneck cancer.

A “therapeutic agent” encompasses both a biological agent such as anantibody, a peptide, a protein, an enzyme or a chemotherapeutic agent.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeErlotinib (TARCEVA®, Genentech/OSI Pharm.), Bortezomib (VELCADE®,Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent(SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate(GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin(Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin(Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, GlaxoSmith Kline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, BayerLabs), and Gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271;Sugen), alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (doxetaxel; Rhone-Poulenc Rorer, Antony, France);chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate;daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; and pharmaceutically acceptable salts,acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors; (v) lipid kinase inhibitors; (vi) antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in aberrant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; a topoisomerase 1 inhibitorsuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and (x) pharmaceutically acceptablesalts, acids and derivatives of any of the above. Other anti-angiogenicagents include MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9(matrix-metalloproteinase 9) inhibitors, COX-II (cyclooxygenase II)inhibitors, and VEGF receptor tyrosine kinase inhibitors. Examples ofsuch useful matrix metalloproteinase inhibitors that can be used incombination with the present compounds/compositions are described in WO96/33172, WO 96/27583, EP 818442, EP 1004578, WO 98/07697, WO 98/03516,WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, EP 606,046, EP931,788, WO 90/05719, WO 99/52910, WO 99/52889, WO 99/29667, WO99/07675, EP 945864, U.S. Pat. No. 5,863,949, U.S. Pat. No. 5,861,510,and EP 780,386, all of which are incorporated herein in their entiretiesby reference. Examples of VEGF receptor tyrosine kinase inhibitorsinclude4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)qu-inazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)-1-quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814), and compounds such asthose disclosed in PCT Publication Nos. WO 97/22596, WO 97/30035, WO97/32856, and WO 98/13354).

Other examples of chemotherapeutic agents that can be used incombination with the present compounds include inhibitors of PI3K(phosphoinositide-3 kinase), such as those reported in Yaguchi et al(2006) Jour. of the Nat. Cancer Inst. 98(8):545-556; U.S. Pat. No.7,173,029; U.S. Pat. No. 7,037,915; U.S. Pat. No. 6,608,056; U.S. Pat.No. 6,608,053; U.S. Pat. No. 6,838,457; U.S. Pat. No. 6,770,641; U.S.Pat. No. 6,653,320; U.S. Pat. No. 6,403,588; WO 2006/046031; WO2006/046035; WO 2006/046040; WO 2007/042806; WO 2007/042810; WO2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO2003/037886; US 2003/149074; WO 2003/035618; WO 2003/034997; US2003/158212; EP 1417976; US 2004/053946; JP 2001247477; JP 08175990; JP08176070; U.S. Pat. No. 6,703,414; and WO 97/15658, all of which areincorporated herein in their entireties by reference. Specific examplesof such PI3K inhibitors include SF-1126 (PI3K inhibitor, SemaforePharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3Kinhibitor, Exelixis, Inc.).

A “metabolite” is a product produced through metabolism in the body of aspecified compound, a derivative thereof, or a conjugate thereof, orsalt thereof. Metabolites of a compound, a derivative thereof, or aconjugate thereof, may be identified using routine techniques known inthe art and their activities determined using tests such as thosedescribed herein. Such products may result for example from theoxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation,esterification, deesterification, enzymatic cleavage, and the like, ofthe administered compound. Accordingly, the invention includesmetabolites of compounds, a derivative thereof, or a conjugate thereof,of the invention, including compounds, a derivative thereof, or aconjugate thereof, produced by a process comprising contacting acompound, a derivative thereof, or a conjugate thereof, of thisinvention with a mammal for a period of time sufficient to yield ametabolic product thereof.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

The term “protecting group” or “protecting moiety” refers to asubstituent that is commonly employed to block or protect a particularfunctionality while reacting other functional groups on the compound, aderivative thereof, or a conjugate thereof. For example, an“amine-protecting group” or an “amino-protecting moiety” is asubstituent attached to an amino group that blocks or protects the aminofunctionality in the compound. Such groups are well known in the art(see for example P. Wuts and T. Greene, 2007, Protective Groups inOrganic Synthesis, Chapter 7, J. Wiley & Sons, NJ) and exemplified bycarbamates such as methyl and ethyl carbamate, FMOC, substituted ethylcarbamates, carbamates cleaved by 1,6-β-elimination (also termed “selfimmolative”), ureas, amides, peptides, alkyl and aryl derivatives.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see P. G. M. Wuts & T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,2007.

The term “leaving group” refers to an group of charged or unchargedmoiety that departs during a substitution or displacement. Such leavinggroups are well known in the art and include, but not limited to,halogens, esters, alkoxy, hydroxyl, tosylates, triflates, mesylates,nitriles, azide, carbamate, disulfides, thioesters, thioethers anddiazonium compounds.

A “linking group” as defined herein refers to a functional group thatcan form a chemical bond with a cell-binding agent. Suitable chemicalbonds are well known in the art and include disulfide bonds, thioetherbonds, acid labile bonds, photolabile bonds, peptidase labile bonds andesterase labile bonds (see for example U.S. Pat. Nos. 5,208,020;5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499;7,368,565; 7,388,026 and 7,414,073). Preferred are disulfide bonds,thioether and peptidase labile bonds. In one embodiment, the linkinggroup is selected from the group consisting of a maleimide, ahaloacetamido, —SH, —SSR^(f), —CH₂SH, —CH(Me)SH, —C(Me)₂SH, —NHR^(g),—CH₂NHR^(g), —NR^(g)NH₂, —COOH, a reactive ester, an amino acid, or apeptide having 2 to 10 amino acids, wherein R^(f) is selected fromphenyl, nitrophenyl (e.g., 2 or 4-nitrophenyl), dinitrophenyl (e.g., 2or 4-nitrophenyl), carboxynitrophenyl (e.g., 3-carboxy-4-nitrophenyl),pyridyl or nitropyridyl (e.g., 4-nitropyridyl) and R^(g) is —H or a C₁₋₄alkyl.

The term “linking moiety” as used herein refers to the remainingchemical moiety of the linking group after the linking group iscovalently linked to a cell-binding agent. For example, when the linkinggroup N-hydroxysuccinimide ester is chemically linked with an aminegroup of the cell-binding agent, the corresponding linking moiety is—C(═O)—. In another example, when the linking group maleimide group ischemically linked to a thio (—SH) group of the cell-binding agent, thelinking moiety is

The “reactive ester” as used herein refers to an ester group having aleaving group that is readily displaced by an amine or a hydroxyl group.Examples of a reactive ester, include, but are not limited to,N-hydroxysuccinimde ester, N-hydroxy sulfosuccinimide ester, nitrophenyl(e.g., 2 or 4-nitrophenyl) ester, dinitrophenyl (e.g.,2,4-dinitrophenyl) ester, sulfo-tetraflurophenyl (e.g.,4-sulfo-2,3,5,6-tetrafluorophenyl)ester and pentafluorophenyl ester.

As used herein, a “bifunctional crosslinking reagent” refers to areagent that possesses two reactive groups; one of which is capable ofreacting with a cell-binding agent while the other one reacts with thecytotoxic compound D-XH to link the two moieties together. Suchbifunctional crosslinkers are well known in the art (see, for example,Isalm and Dent in Bioconjugation chapter 5, p 218-363, GrovesDictionaries Inc. New York, 1999). For example, bifunctionalcrosslinking agents that enable linkage via a thioether bond includeN-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) tointroduce maleimido groups. Other bifunctional crosslinking agents thatintroduce maleimido groups on to a cell binding agent are well known inthe art (see US Patent Applications 2008/0050310, 20050169933, availablefrom Pierce Biotechnology Inc. P.O. Box 117, Rockland, Ill. 61105, USA)and include, but not limited to, N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC),N-(α-maleimidoacetoxy)-succinimide ester (AMAS), κ-maleimidoundecanoicacid N-succinimidyl ester (KMUA), N-(β-maleimidopropyloxy)succinimideester (BMPS), γ-maleimidobutyric acid N-succinimidyl ester (GMBS),ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-(α-maleimidoacetoxy)-succinimide ester (AMAS),succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl4-(p-maleimidophenyl)-butyrate (SMPB), andN-(p-maleimidophenyl)isocyanate (PMPI). Cross-linking reagentscomprising a haloacetyl-based moiety includeN-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl3-(bromoacetamido)propionate (SBAP),succinimidyl-(4-vinylsulfonyl)benzoate (SBSV),bis-maleimidopolyethyleneglycol (BMPEO), BM(PEO)₂, BM(PEO)₃,5-maleimidovaleric acid NHS, HBVS, 4-(4-N-maleimidophenyl)-butyric acidhydrazide.HCl (MPBH), Succinimidyl-(4-vinylsulfonyl)benzoate (SVSB),dithiobis-maleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH),bis-maleimidoethane (BMOE), sulfosuccinimidyl4-(N-maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC),sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-S TAB),m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS),N-(γ-maleimidobutryloxy)sulfosuccinimde ester (sulfo-GMBS),N-(ε-maleimidocaproyloxy)sulfosuccimido ester (sulfo-EMCS),N-κ-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS),sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB), CX1-1,sulfo-Mal and PEG_(n)-Mal. Preferably, the bifunctional crosslinkingreagent is SMCC.

wherein n is 2 to 24. Preferably, for PEG_(n)-Mal, n is 2 to 10. Morepreferably, n is 2, 4, 6 or 8.

The term “therapeutically effective amount” means that amount of activecompound or conjugate that elicits the desired biological response in asubject. Such response includes alleviation of the symptoms of thedisease or disorder being treated, prevention, inhibition or a delay inthe recurrence of symptom of the disease or of the disease itself, anincrease in the longevity of the subject compared with the absence ofthe treatment, or prevention, inhibition or delay in the progression ofsymptom of the disease or of the disease itself. Determination of theeffective amount is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.Toxicity and therapeutic efficacy of compound I can be determined bystandard pharmaceutical procedures in cell cultures and in experimentalanimals. The effective amount of compound or conjugate of the presentinvention or other therapeutic agent to be administered to a subjectwill depend on the stage, category and status of the multiple myelomaand characteristics of the subject, such as general health, age, sex,body weight and drug tolerance. The effective amount of compound orconjugate of the present invention or other therapeutic agent to beadministered will also depend on administration route and dosage form.Dosage amount and interval can be adjusted individually to provideplasma levels of the active compound that are sufficient to maintaindesired therapeutic effects.

Cytotoxic Compounds

The present invention is directed to cytotoxic compounds (e.g.,compounds of structural formulas(I′), (I) and (II)) and conjugates(e.g., conjugates represented by structural formula (III′), (III) or(IV)) described herein.

In one embodiment, for structural formulas (I′), (I), (II), (III′),(III) or (IV), the definitions for the variables depicted therein are asdefined below:

1. D in structural formulas (I′), (I), (II), (III′), (III) or (IV) is acytotoxic agent. In a preferred embodiment, D is a maytansinoid. In amore preferred embodiment, D is represented by the following structuralformula:

wherein:

-   -   Y₁ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂—,    -   R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, an        alkyl, an alkenyl, a cycloalkyl, a heteroaryl, a heterocyclyl,        or an aryl; and    -   l, m and n are each independently 0 or an integer from 1 to 5.

In another preferred embodiment, D is represented by structural formula(Al), wherein R₁ and R₂ are each independently H, C₁₋₄ alkyl or C₁₋₄alkyl substituted with one to six halogens. More preferably, R₁ and R₂are each independently H, methyl, —CF₃ or —CCl₃. Even more preferably,R₁ and R₂ are both H or methyl.

In another preferred embodiment, D is represented by structural formula(Al), wherein l and m are 0, n is 1, R₁, R₂, R₃ and R₄ are all H.

In another preferred embodiment, D is represented by structural formula(Al), wherein n is 0, l and m are both 1, R₅, R₆, R₇ and R₈ are all H,R₁ and R₂ are both methyl.

In yet another preferred embodiment, D is represented by structuralformula (Al), wherein n is 0, l and m are both 1, R₁, R₅, R₆, R₇ and R₈are all H, and R₂ is methyl

2. X in structural formulas (I), (II), (III) and (IV) is S or Se. In oneembodiment, X is S. In another embodiment, X is Se.

3. R and R′ in structural formulas (I) and (III) are each independentlyselected from the group consisting of —H, an alkyl, a cycloalkyl,—OR^(a), and —NR^(b)R^(c), wherein R^(a), R^(b) and R^(c) are eachindependently H or an alkyl. Preferably, one of R and R′ is H, and theother is H, a C₁₋₄ alkyl, —OR^(a) or —NR^(b)R^(c), wherein R^(a), R^(b)and R^(c) are each independently H or C₁₋₄ alkyl. More preferably, R andR′ are both H.

4. p in structural formulas (I) and (III) is 0, 1 or 2. Preferably, p is0.

5. Y″ in structural formulas (I′) and (III′) is a spacer. In oneembodiment, when p is O, —Y″M is the non-maleimide moiety of amaleimide-containing bifunctional crosslinking reagent. In anotherembodiment, Y″ is an alkylene, a polyethylene glycol unit represented by—(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or —R^(d)—W—R^(e)′—, wherein:

-   -   n is an integer from 1 to 24;    -   E is a cycloalkyl, a heterocyclyl, an aryl or a heteroaryl,    -   W is —C(═O)NH—, —NHC(═O)—, —(C═O)O— or —O(C═O)—,    -   R^(d) is absent or an alkyl; and    -   R^(e)′ is an alkyl or a polyethylene glycol unit represented by        —(CH₂—CH₂—O)_(n)—R^(d)—.

In one embodiment, Y in structural formulas (I) and (III) is analkylene, a polyethylene glycol unit represented by—(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or —R^(d)—W—R^(e)—, wherein:

-   -   n is an integer from 1 to 24;    -   E is a cycloalkyl, a heterocyclyl, an aryl or a heteroaryl,    -   W is —C(═O)NH—, —NHC(═O)—, —(C═O)O— or —O(C═O)—,    -   R^(d) is absent or an alkyl; and    -   R^(e) is an alkyl.

Preferably, Y is a C₁₋₆ alkylene, a polyethylene glycol unit representedby —(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or —R^(d)—W—R^(e)—, wherein R^(d)is absent or a C₁₋₄ alkyl; R^(e) is a C₁₋₄ alkyl; W is —C(═O)NH—; E iscyclohexyl and n is an integer from 2 to 8.

In another preferred embodiment, Y is

5. M in structural formula (I′), (I) and (II) is a linking group thatcan react with a cell-binding agent to form a covalent bond. Preferably,M is a maleimide, a haloacetamido, —SH, —SSR^(f), —CH₂SH, —CH(Me)SH,—C(Me)₂SH, —NHR^(g), —CH₂NHR^(g), —NR^(g)NH₂, —COOH, a reactive ester,an amino acid or a peptide comprising 2 to 10 amino acids, wherein R^(f)is selected from phenyl, nitrophenyl (e.g., 2 or 4-nitrophenyl),dinitrophenyl (e.g., 2 or 4-nitrophenyl), carboxynitrophenyl (e.g.,3-carboxy-4-nitrophenyl), pyridyl or nitropyridyl (e.g., 4-nitropyridyl)and R^(g) is —H or a C₁₋₄ alkyl.

In a preferred embodiment, M is a maleimide, haloacetamido,N-hydroxysuccinimde ester, N-hydroxy sulfosuccinimide ester, nitrophenyl(e.g., 2 or 4-nitrophenyl)ester, dinitrophenyl (e.g., 2,4-dinitrophenyl)ester, sulfo-tetraflurophenyl (e.g.,4-sulfo-2,3,5,6-tetrafluorophenyl)ester or pentafluorophenyl ester. Morepreferably, M is N-hydroxysuccinimide ester.

6. M′ in structural formulas (III′), (III) and (IV) is a linking moietythat covalently linked to the cell-binding agent. Preferably, M′ is—C(═O)—,

—NH—C(═O)—CH₂—, —S—, —CH₂—S—, —CH(Me)-S—, —C(Me₂)-S—, —NR^(g)—,—CH₂NR^(g)—, or —NR^(g)N═, wherein R^(g) is —H or a C₁₋₄ alkyl. Morepreferably, M′ is —C(═O)—.

7. For structural formulas (II) and (IV), Y′ is —SO₂—, aryl, pyridyl or—C(═)—; Z is an alkyl, a cycloalkyl, a heterocyclyl, an aryl, aheteroaryl, or —N(R^(b))—R^(d)—; wherein R^(b) is H or a C₁₋₄ alkyl; andR^(d) is a C₁₋₄ alkyl.

In a preferred embodiment, Y′ is —SO₂—; and Z is optionally substitutedphenyl. More preferably, Z is unsubstituted phenyl.

In another preferred embodiment, Y′ is phenyl or pyridyl, morepreferably, Y′ is pyridyl and Y′ is linked with Z through the pyridylnitrogen atom. Z is an alkyl. Preferably, Z is a C₁₋₄ alkyl.

In yet another preferred embodiment, Y′ is —C(═O)—, and Z is—N(R^(b))—R^(d)—, wherein R^(b) is H and R^(d) is —CH₂—CH₂—.

In a first embodiment, the present invention is directed to a compoundof structural formula (I′) or (I):

wherein the definitions for each variable are described above.

In a second embodiment, for the compound of structural formula (I′) or(I):

D is represented by structural formula (Al):

Y₁ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂—,

R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, an alkyl, analkenyl, a cycloalkyl, a heteroaryl, a heterocyclyl, or an aryl; and

l, m and n are each independently 0 or an integer from 1 to 5;

p is 0;

X is S; and the remainder of the variables are as described above forstructural formula (I).

In a third embodiment, for compound of structural formula (I′) or (I):

D is represented by structural formula (A2):

p is 0;

X is S; and the remainder of the variables are as described above forstructural formula (I).

In a fourth embodiment, for compound of structural formula (I′) or (I):

D is represented by structural formula (A3):

p is 0;

X is S; and the remainder of the variables are as described above forstructural formula (I).

In a fifth embodiment, for compound of structural formula (I′) describedin the second, third or fourth embodiment, when p is 0, —Y″M is thenon-maleimide moiety of a maleimide-containing bifunctional crosslinkingreagent. In another embodiment, for compound of structural formula (I′),Y″ is an alkylene, a polyethylene glycol unit represented by—(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or —R^(d)—W—R^(e)′—, wherein:

-   -   n is an integer from 1 to 24;    -   E is a cycloalkyl, a heterocyclyl, an aryl or a heteroaryl,    -   W is —C(═O)NH—, —NHC(═O)—, —(C═O)O— or —O(C═O)—,    -   R^(d) is absent or an alkyl; and    -   R^(e)′ is an alkyl or —(CH₂—CH₂—O)_(n)—R^(d)—.

Preferably, Y″ is —R^(d)—W—R^(e)′—, wherein R^(d) is a C₁₋₄ alkyl, W is—C(═O)NH— or —NHC(═O)—, and R^(e)′ is —(CH₂—CH₂—O)_(n)—R^(d)—. Even morepreferably, n is an integer from 2 to 10.

In another embodiment, for compound of structural formula (I) describedin the second, third or fourth embodiment:

Y is a C₁₋₆ alkylene, a polyethylene glycol unit represented by—(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or —R^(d)—W—R^(e)—, wherein R^(d) isabsent or a C₁₋₄ alkyl; R^(e) is a C₁₋₄ alkyl; W is —C(═O)NH—; E iscyclohexyl and n is an integer from 2 to 8; and

M is a maleimide, a haloacetamido, —SH, —SSR^(f), —CH₂SH, —CH(Me)SH,—C(Me)₂SH, —NHR^(g), —CH₂NHR^(g), —NR^(g)NH₂, —COOH, a reactive ester,an amino acid, a peptide comprising 2 to 10 amino acids, wherein R^(f)is selected from phenyl, nitrophenyl (e.g., 2 or 4-nitrophenyl),dinitrophenyl (e.g., 2 or 4-nitrophenyl), carboxynitrophenyl (e.g.,3-carboxy-4-nitrophenyl), pyridyl or nitropyridyl (e.g., 4-nitropyridyl)and R^(g) is —H or a C₁₋₄ alkyl.

Preferably, Y is

Even more preferably, Y is

and M is N-hydroxysuccinimide ester.

In a sixth embodiment, the present invention is directed to a compoundrepresented by structural formula (II):

wherein the variables are as described above for structural formula(II).

In a seventh embodiment, for compound of structural formula (II):

D is represented by structural formula (A1):

Y₁ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂—,

R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, an alkyl, analkenyl, a cycloalkyl, a heteroaryl, a heterocyclyl, or an aryl;

l, m and n are each independently 0 or an integer from 1 to 5;

X is S; and the remainder of the variables are as described instructural formula (II).

In a preferred embodiment, Y′ is SO₂ and Z is an optionally substitutedphenyl. The remainder of the variables is as described above in theseventh embodiment. More preferably, Z is an unsubstituted phenyl.

In another preferred embodiment, Y′ is pyridyl or phenyl; Z is an alkyl;more preferably, Y′ is pyridyl and Y′ is linked with Z through thepyridyl nitrogen. The remainder of the variables is as described abovein the seventh embodiment. More preferably, Z is a C₁₋₄ alkyl.

In another preferred embodiment, Y′ is —C(═O)—; and Z is—N(R^(b))—R^(d)—, wherein R^(b) is H or an alkyl; and R^(d) is an alkyl.The remainder of the variables is as described above in the seventhembodiment. More preferably, R^(b) is H and R^(d) is a C₁₋₄ alkyl. Evenmore preferably, R^(b) is H and R^(d) is —CH₂—CH₂—.

In a eighth embodiment, for compound of structural formula (II):

D is represented by structural formula (A2):

X is S; and the remainder of the variables are as described instructural formula (II).

In a preferred embodiment, Y′ is SO₂ and Z is an optionally substitutedphenyl. The remainder of the variables is as described above in theeighth embodiment. More preferably, Z is an unsubstituted phenyl.

In another preferred embodiment, Y′ is phenyl or pyridyl; Z is an alkyl;more preferably, Y′ is pyridyl and Y′ is linked with Z through thepyridyl nitrogen. The remainder of the variables is as described abovein the eighth embodiment. More preferably, Z is a C₁₋₄ alkyl.

In another preferred embodiment, Y′ is —C(═O)—; and Z is—N(R^(b))—R^(d)—, wherein R^(b) is H or an alkyl; and R^(d) is an alkyl.The remainder of the variables is as described above in the eighthembodiment. More preferably, R^(b) is H and R^(d) is a C₁₋₄ alkyl. Evenmore preferably, R^(b) is H and R^(d) is —CH₂—CH₂—.

In a ninth embodiment, for compound of structural formula (II):

D is represented by structural formula (A3):

X is S; and the remainder of the variables are as described instructural formula (II).

In a preferred embodiment, Y′ is SO₂ and Z is an optionally substitutedphenyl. The remainder of the variables is as described above in theninth embodiment. More preferably, Z is an unsubstituted phenyl.

In another preferred embodiment, Y′ is phenyl or pyridyl; Z is an alkyl;and more preferably, Y′ is pyridyl and Y′ is linked with Z through thepyridyl nitrogen. The remainder of the variables is as described abovein the ninth embodiment. More preferably, Z is a C₁₋₄ alkyl.

In another preferred embodiment, Y′ is —C(═O)—; and Z is—N(R^(b))—R^(d)—, wherein R^(b) is H or an alkyl; and R^(d) is an alkyl.The remainder of the variables is as described above in the ninthembodiment. More preferably, R^(b) is H and R^(d) is a C₁₋₄ alkyl. Evenmore preferably, R^(b) is H and R^(d) is —CH₂—CH₂—.

Cell-Binding Agents

The effectiveness of the conjugates of the invention as therapeuticagents depends on the careful selection of an appropriate cell-bindingagent. Cell-binding agents may be of any kind presently known, or thatbecome known and includes peptides and non-peptides. Generally, thesecan be antibodies (especially monoclonal antibodies), lymphokines,hormones, growth factors, vitamins, nutrient-transport molecules (suchas transferrin), or any other cell-binding molecule or substance.

More specific examples of cell-binding agents that can be used include:

polyclonal antibodies;

monoclonal antibodies;

fragments of antibodies such as Fab, Fab′, and F(ab′)₂, Fv, minibodies,diabodies, tribodies, tetrabodies, nanobodies, probodies, domain bodies,unibodies and the like (Parham, J. Immunol. 131:2895-2902 (1983); Springet al. J. Immunol. 113:470-478 (1974); Nisonoff et al. Arch. Biochem.Biophys. 89:230-244 (1960), Kim et al., Mol, Cancer Ther., 7: 2486-2497(2008), Carter, Nature Revs., 6: 343-357 (2006), R. Kontermann & S.Dubel, 2001 Antibody Engineering, Springer-Verlag, Heidelberg-New York);

bispecific antibodies (Morrison, S L Nature biotechnology 25 (11):1233-4 (2007));

ankyrin repeat proteins (DARPins; Zahnd et al., J. Biol. Chem., 281, 46,35167-35175, (2006); Binz, H. K., Amstutz, P. & Pluckthun, A. (2005)Nature Biotechnology, 23, 1257-1268) or ankyrin-like repeats proteins orsynthetic peptides described, for example, in U.S. Patent PublicationNumber 20070238667; U.S. Pat. No. 7,101,675; and WO/2007/147213;WO/2007/062466)

interferons (e.g. .alpha., .beta., .gamma.);

lymphokines such as IL-2, IL-3, IL-4, IL-6;

hormones such as insulin, TRH (thyrotropin releasing hormone), MSH(melanocyte-stimulating hormone), steroid hormones, such as androgensand estrogens;

growth factors and colony-stimulating factors such as EGF, TGF-alpha,FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158(1984));

transferrin (O'Keefe et al. J. Biol. Chem. 260:932-937 (1985)); and

vitamins, such as folate.

Monoclonal antibody techniques allow for the production of extremelyspecific cell-binding agents in the form of specific monoclonalantibodies. Particularly well known in the art are techniques forcreating monoclonal antibodies produced by immunizing mice, rats,hamsters or any other mammal with the antigen of interest such as theintact target cell, antigens isolated from the target cell, whole virus,attenuated whole virus, and viral proteins such as viral coat proteins.Sensitized human cells can also be used. Another method of creatingmonoclonal antibodies is the use of phage libraries of scFv (singlechain variable region), specifically human scFv (see e.g., Griffiths etal., U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al., WO92/01047; Liming et al., WO 99/06587). In addition, resurfacedantibodies disclosed in U.S. Pat. No. 5,639,641 may also be used, as maychimeric antiobodies and humanized antibodies. Selection of theappropriate cell-binding agent is a matter of choice that depends uponthe particular cell population that is to be targeted, but in generalhuman monoclonal antibodies are preferred if an appropriate one isavailable.

For example, the monoclonal antibody MY9 is a murine IgG₁ antibody thatbinds specifically to the CD33 Antigen {J. D. Griffin et al 8 LeukemiaRes., 521 (1984)} and can be used if the target cells express CD33 as inthe disease of acute myelogenous leukemia (AML). The cell-binding agentmay be any compound that can bind a cell, either in a specific ornon-specific manner. Generally, these can be antibodies (especiallymonoclonal antibodies and antibody fragments), interferons, lymphokines,hormones, growth factors, vitamins, nutrient-transport molecules (suchas transferrin), or any other cell-binding molecule or substance.

Where the cell-binding agent is an antibody, it binds to an antigen thatis a polypeptide and may be a transmembrane molecule (e.g. receptor) ora ligand such as a growth factor. Exemplary antigens include moleculessuch as renin; a growth hormone, including human growth hormone andbovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor vmc, factor IX, tissue factor (TF), and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial natriureticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA); bombesin;thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); a serum albumin, such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins, EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2,EphA receptors, EphB receptors, folate receptor, FOLR1, mesothelin,cripto, alpha_(v)beta₆, integrins, VEGF, VEGFR, EGFR, transferrinreceptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2,CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26,CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59,CD70, CD79, CD80. CD81, CD103, CD105, CD134, CD137, CD138, CD152 or anantibody which binds to one or more tumor-associated antigens orcell-surface receptors disclosed in US Publication No. 20080171040 or USPublication No. 20080305044 and are incorporated in their entirety byreference; erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon, such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe HIV envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; endoglin, c-Met, 1GF1R, PSGR, NGEP, PSMA, PSCA, LGR5,B7H4, TAG72 (tumor-associated glycoprotein 72), and fragments of any ofthe above-listed polypeptides.

Additionally, GM-CSF, which binds to myeloid cells can be used as acell-binding agent to diseased cells from acute myelogenous leukemia.IL-2 which binds to activated T-cells can be used for prevention oftransplant graft rejection, for therapy and prevention ofgraft-versus-host disease, and for treatment of acute T-cell leukemia.MSH, which binds to melanocytes, can be used for the treatment ofmelanoma, as can antibodies directed towards melanomas. Folic acid canbe used to target the folate receptor expressed on ovarian and othertumors. Epidermal growth factor can be used to target squamous cancerssuch as lung and head and neck. Somatostatin can be used to targetneuroblastomas and other tumor types.

Cancers of the breast and testes can be successfully targeted withestrogen (or estrogen analogues) or androgen (or androgen analogues)respectively as cell-binding agents.

In one embodiment, the antibody is selected from the group consisting ofhuN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab,rituximab, huDS6, anti-mesothelin antibodies described in WO 2010/124797(such as MF-T), anti-cripto antibodies described in US PatentApplication Publication 2010/0093980 (such as huB3F6), anti-CD138antibodies described in US Patent Application Publication 2007/0183971(such as huB-B4), anti-EGFRvIII antibodies described U.S. Pat. Nos.7,736,644 and 7,628,986 and US Application Publication Nos.2010/0111979, 2009/0240038, 2009/0175887, 2009/0156790 and 2009/0155282,humanized EphA2 antibodies described in PCT/IB2010/054417 andPCT/IB2010/054422 (such as 2H11R35R74); anti-CD38 antibodies describedin WO2008/047242 (such as hu38SB19), anti-folate receptor antibodiesdescribed in U.S. Provisional Application Nos. 61/307,797, 61/346,595and 61/413,172, and U.S. application Ser. No. 13/033,723 (e.g.,huMov19); anti-IGF1R antibodies described in U.S. Pat. Nos. 5,958,872and 6,596,743; anti-CD37 antibodies described in U.S. application Ser.No. 13/045,693, filed on Mar. 11, 2011 (e.g., huCD37-3); anti-integrinα_(v)β₆ antibodies described in U.S. Application Publication No.2006/0127407 (e.g., CNTO95); and anti-Her3 antibodies described in U.S.Provisional Application Nos. 61/370,701.

Particularly preferred antibodies are humanized monoclonal antibodiesdescribed herein. Examples include, but are not limited to, huN901,huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, CNTO95,huDS6, and rituximab (see, e.g., U.S. Pat. Nos. 5,639,641 and 5,665,357,U.S. Provisional Patent Application No. 60/424,332 (which is related toU.S. Pat. No. 7,557,189), International (PCT) Patent ApplicationPublication WO 02/16401, Pedersen et al., supra, Roguska et al., supra,Liu et al., supra, Nadler et al., supra, Colomer et al., Cancer Invest.,19: 49-56 (2001), Heider et al., Eur. J. Cancer, 31A: 2385-2391 (1995),Welt et al., J. Clin. Oncol., 12: 1193-1203 (1994), and Maloney et al.,Blood, 90: 2188-2195 (1997)). Other humanized monoclonal antibodies areknown in the art and can be used in connection with the invention.

In one embodiment, the anti-folate antibody is a humanized antibody orantigen binding fragment thereof that specifically binds a human folatereceptor 1, wherein the antibody comprises: (a) a heavy chain CDR1comprising GYFMN; a heavy chain CDR2 comprisingRIHPYDGDTFYNQXaa₁FXaa₂Xaa₃; and a heavy chain CDR3 comprising YDGSRAMDY;and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH; a light chainCDR2 comprising RASNLEA; and a light chain CDR3 comprising QQSREYPYT;wherein Xaa₁ is selected from K, Q, H, and R; Xaa₂ is selected from Q,H, N, and R; and Xaa₃ is selected from G, E, T, S, A, and V. Preferably,the heavy chain CDR2 sequence comprises RIHPYDGDTFYNQKFQG.

In another embodiment, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof that specifically binds thehuman folate receptor 1 comprising the heavy chain having the amino acidsequence of QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK.

In another embodiment, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof encoded by the plasmid DNAdeposited with the ATCC on Apr. 7, 2010 and having ATCC deposit nos.PTA-10772 and PTA-10773 or 10774.

In another embodiment, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof comprising a heavy chainvariable domain at least about 90%, 95%, 99% or 100% identical toQVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDY WGQGTTVTVSS, and alight chain variable domain at least about 90%, 95%, 99% or 100%identical to DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIK R; orDIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIK R.

Cell-Binding Agent-Cytotoxic Compound Conjugates

The present invention also provides cell-binding agent-cytotoxiccompound conjugates comprising a cell-binding agent linked to one ormore cytotoxic compounds of the present invention via a variety oflinkers, including, but not limited to, disulfide linkers, thioetherlinkers, amide bonded linkers, peptidase-labile linkers, acid-labilelinkers, esterase-labile linkers. Representative conjugates of theinvention are antibody/cytotoxic compound, antibody fragment/cytotoxiccompound, epidermal growth factor (EGF)/cytotoxic compound, melanocytestimulating hormone (MSH)/cytotoxic compound, thyroid stimulatinghormone (TSH)/cytotoxic compound, somatostatin/cytotoxic compound,folate/cytotoxic compound, estrogen/cytotoxic compound, estrogenanalogue/cytotoxic compound, androgen/cytotoxic compound, and androgenanalogue/cytotoxic compound.

In one embodiment, the present invention provides conjugates comprisinga cell-binding agent chemically linked to a compound of the presentinvention (e.g., compounds of formula (I′), (I) or (II)).

In one embodiment, the cell-binding agent can linked to the compoundthrough an amine group, a thio group or a carboxy group.

In a tenth embodiment, the present invention is directed to a conjugaterepresented by structural formula (III′) or (III):

wherein values or preferred values for the variables are as describedabove for structural formula (III′) or (III).

In a eleventh embodiment, for the conjugate of structural formula (III):

D is represented by structural formula (A1):

Y₁ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂—,

R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, an alkyl, analkenyl, a cycloalkyl, a heteroaryl, a heterocyclyl, or an aryl; and

l, m and n are each independently 0 or an integer from 1 to 5;

p is 0;

X is S; and the remainder of the variables are as described above forstructural formula (III).

In a twelfth embodiment, for the conjugate of structural formula (III):

D is represented by structural formula (A2):

p is 0;

X is S; and values and preferred values for the remainder of thevariables is as described above for structural formula (III).

In a thirteen embodiment, for the conjugate of structural formula (III):

D is represented by structural formula (A3):

p is 0;

X is S; and values and preferred values for the remainder of thevariables are as described above for structural formula (III).

In a fourteenth embodiment, for conjugates of structural formula (III′)in the eleventh, twelfth or thirteen embodiment, when p is 0, —Y″ is thenon-maleimide and non-linking group moiety of a maleimide-containingbifunctional crosslinking reagent. In another embodiment, for compoundof structural formula (III′), Y″ is an alkylene, a polyethylene glycolunit represented by —(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or—R^(d)—W—^(e)′—, wherein:

-   -   n is an integer from 1 to 24;    -   E is a cycloalkyl, a heterocyclyl, an aryl or a heteroaryl,    -   W is —C(═O)NH—, —NHC(═O)—, —(C═O)O— or —O(C═O)—,    -   R^(d) is absent or an alkyl; and    -   R^(e)′ is an alkyl or —(CH₂—CH₂—O)_(n)—R^(d)—.

Preferably, Y is —R^(d)—W—R^(e)′—, wherein R^(d) is a C₁₋₄ alkyl, W is—C(═O)NH— or —NHC(═O)—, and R^(e)′ is —(CH₂—CH₂—O)_(n)—R^(d)—. Even morepreferably, n is an integer from 2 to 10.

Even more preferably, Y″ in structural formula (III′) is represented bythe following structural formula:

Preferably, n is an integer from 2 to 10. Even more preferably, n is 2,4 or 8.

In another embodiment, for the conjugates of structural formula (III)described in the eleventh, twelfth or thirteen embodiment,

Y is a C₁₋₆ alkylene, a polyethylene glycol unit represented by—(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or —R^(d)—W—R^(e)—, wherein R^(d) isabsent or a C₁₋₄ alkyl; R^(e) is a C₁₋₄ alkyl; W is —C(═O)NH—; E iscyclohexyl and n is an integer from 2 to 8; and

M′ is —C(═O)—,

—NH—C(═O)—CH₂—, —S—, —CH₂—S—, —CH(Me)-S—, —C(Me₂)-S—, —NR^(g)—,—CH₂NR^(g)—, or —NR^(g)N═, wherein R^(g) is —H or a C₁₋₄ alkyl.

Preferably, Y is

Even more preferably, Y is

and M is —C(═O)—. Even more preferably, the CBA is linked to M throughan amine (e.g. CBA-NH₂) group.

In a fifteenth embodiment, the present invention is directed to aconjugate represented by structural formula (IV):

wherein the values and preferred values for the variables are asdescribed above for structural formula (IV).

In a sixteenth embodiment, for the conjugate of structural formula (IV):

D is represented by structural formula (A1):

Y₁ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂—,

R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, an alkyl, analkenyl, a cycloalkyl, a heteroaryl, a heterocyclyl, or an aryl;

l, m and n are each independently 0 or an integer from 1 to 5;

X is S; and values and preferred values for the remainder of thevariables is as described in structural formula (IV).

In a preferred embodiment, Y′ is SO₂ and Z is an optionally substitutedphenyl. The remainder of the variables is as described above in thesixteenth embodiment. More preferably, Z is an unsubstituted phenyl.

In another preferred embodiment, Y′ is pheny or pyridyl; Z is an alkyl;and preferably, Y′ is pyridyl and Y′ is linked with Z through thepyridyl nitrogen. The remainder of the variables is as described abovein the sixteenth embodiment. More preferably, Z is a C₁₋₄ alkyl.

In another preferred embodiment, Y′ is —C(═O)—; and Z is—N(R^(b))—R^(d)—, wherein R^(b) is H or an alkyl; and R^(d) is an alkyl.The remainder of the variables is as described above in the sixteenthembodiment. More preferably, R^(b) is H and R^(d) is a C₁₋₄ alkyl. Evenmore preferably, R^(b) is H and R^(d) is —CH₂—CH₂—.

In a seventeenth embodiment, for the conjugates of structural formula(IV):

D is represented by structural formula (A2):

X is S; and values and preferred values for the remainder of thevariables are as described in structural formula (II).

In a preferred embodiment, Y′ is SO₂ and Z is an optionally substitutedphenyl. The remainder of the variables is as described above in theseventeenth embodiment. More preferably, Z is an unsubstituted phenyl.

In another preferred embodiment, Y′ is phenyl or pyridyl; Z is an alkyl;and more preferably, Y′ is pyridyl and Y′ is linked with Z through thepyridyl nitrogen. The remainder of the variables is as described abovein the seventeenth embodiment. More preferably, Z is a C₁₋₄ alkyl.

In another preferred embodiment, Y′ is —C(═O)—; and Z is—N(R^(b))—R^(d)—, wherein R^(b) is H or an alkyl; and R^(d) is an alkyl.The remainder of the variables is as described above in the seventeenthembodiment. More preferably, R^(b) is H and R^(d) is a C₁₋₄ alkyl. Evenmore preferably, R^(b) is H and R^(d) is —CH₂—CH₂—.

In a eighteenth embodiment, for the conjugates for formula (IV):

D is represented by structural formula (A3):

X is S; and values and preferred values for the remainder of thevariables is as described in structural formula (II).

In a preferred embodiment, Y′ is SO₂ and Z is an optionally substitutedphenyl. The remainder of the variables is as described above in theeighteenth embodiment. More preferably, Z is an unsubstituted phenyl.

In another preferred embodiment, Y′ is phenyl or pyridyl; Z is an alkyl;and more preferably, Y′ is pyridyl and Y′ is linked with Z through thepyridyl nitrogen. The remainder of the variables is as described abovein the eighteenth embodiment. More preferably, Z is a C₁₋₄ alkyl.

In another preferred embodiment, Y′ is —C(═O)—; and Z is—N(R^(b))—R^(d)—, wherein R^(b) is H or an alkyl; and R^(d) is an alkyl.The remainder of the variables is as described above in the eighteenthembodiment. More preferably, R^(b) is H and R^(d) is a C₁₋₄ alkyl. Evenmore preferably, R^(b) is H and R^(d) is —CH₂—CH₂—.

In a nineteenth embodiment, the conjugate of the present invention isrepresented by the following structural formula:

wherein:

q is an integer from 1 to 20;

n is an integer from 2 to 24;

R₁ and R₂ are H; and n′ is 1; or

R₁ and R₂ are methyl; and n′ is 2.

In another embodiment, the present invention is directed to acell-binding agent-cytotoxic agent conjugate comprising a cell-bindingagent covalently linked to a cytotoxic agent through a sulfoxide linker,wherein the sulfoxide linker is derived from oxidation of a —SH group onthe cell-binding agent. In one embodiment, —SH is from a cysteineresidue of the cell-binding agent.

In another embodiment, the conjugate is represented by the followingstructural formula:

wherein:

CBA is a cell-binding agent;

D is a cytotoxic agent; and

L is a linker group that covalent links the cytotoxic agent with thecell-binding agent.

More specifically, the sulfoxide group

in structural formula (X) is derived from oxidation of a —SH group of acysteine residue on the cell-binding agent.

In one embodiment, L is a suitable linker group known in the art. Forexample, L is a linker group comprising a disulfide group, a thioethergroup, an acid labile group, a photolabile group, a peptidase labilegroup, an esterase labile group, or a combination thereof.

In one embodiment, L is derived from a bifunctional crosslinking reagentcomprising a maleimide at one end of the bifunctional crosslinkingreagent, wherein L represents the remaining portion of the bifunctionalcrosslinking reagent without the terminal maleimide group. Thebifunctional crosslinking reagent is a reagent that possesses tworeactive groups, one of which is a maleimide group that reacts with —SHgroup on the cell-binding agent; while the other one reacts with thecytotoxic agent D. More specifically, the bifunctional crosslinkingreagent can be a bifunctional crosslinking reagent described herein. Forexample, when the bifunctional crosslinking reagent is1,4-bis-maleimidobutane (BMB) and D is a thiol-containing cytotoxicagent, then L is represented by the following structural formula:

In one embodiment, for the conjugate of structural formula (X), D is amaytansinoid. More preferably, D is a maytansinoid represented bystructural formula (A1), (A2) or (A3) described above.

In another embodiment, D is auristatin.

Production of the Cytotoxic Compounds and the Cell-BindingAgent-Cytotoxic Compound Conjugates

The cytototoxic compounds of the present invention (e.g., compounds ofstructural formula (I′), (I) and (II)) can be prepared by any suitablemethods. In one embodiment, the compounds of structural formula (I′) or(I) can be prepared by reacting a compound of structural formula (V′) or(V) with an oxidant. Similarly, the compounds of structural formula (II)can be prepared by reacting a compound of structural formula (VI) withan oxidant.

Any suitable oxidant capable of oxidizing a thiol ether group (—S—) to asulfoxide group (—S(O)—) can be used. Examples of oxidants include, butare not limited to, dimethyldioxirane, NaIO₄, t-BuOCl, dioxiranes,calcium hypochlorite Ca(OCl)₂, sodium chlorite NaClO₂, HNO₃ (optionallyin the presence of a catalyst, such as a AuCl₄ ⁻ catalyst), O₂ (in thepresence of a catalyst, such as a ceric ammonium nitrate catalyst), acylnitrites, sodium perborate, organic peroxides (e.g., ROOH, wherein R isa C₁₋₆ alkyl), and H₂O₂ (optionally in the presence of a catalyst, suchas a bis(acetylacetonato)oxovanadium catalyst).

The oxidation reaction can be carried out in a solvent. The solvent canbe an organic solvent or mixture of one or more organic solvents, or amixture of one or more organic solvents with water or an aqueous buffer.Examples of organic solvents can be used include, but are not limited,to dimethylacetimide (DMA), methanol, ethanol, methylene chloride,acetone, DMF, DMSO, ethyl acetate, dioxane, and acetonitrile.

In one embodiment, the oxidation reaction can be carried out in amixture of dimethylacetimide (DMA) and a succinate buffer. Preferably,the succinate buffer has a pH of about 5. More preferably, the reactioncan be carried out in a 60:40 (v:v) mixture of DMA and succinate bufferhaving a pH about 5.

The oxidation reaction can be carried out at a suitable temperature. Inone embodiment, the reaction is carried out at room temperature (about20-25° C.). Alternatively, the oxidation reaction can be carried out ata low temperature, for example, at about −20° C. to about 15° C. In apreferred embodiment, the oxidation reaction is carried out at about 4°C. In another alternative, the reaction can be carried out at anelevated temperature, for example, higher than about 30° C.

Compounds of formula (V′), (V) and (VI) can be prepared by any suitablemethods. In one embodiment, compounds of formula (V′), (V) or (VI) canbe prepared by reacting compound D-XH with a bifunctional crosslinkingreagent.

In one embodiment, the bifunctional crosslinking reagent comprises amaleimide group (e.g., SMCC, AMAS, BMPS, GMBS, EMCS, SVSB andPEG_(n)-Mal, etc.) and the compound of structural formula (V′) or (V)can be prepared according to scheme 2.

The reaction of D-XH and the bifunctional crosslinking reagent can becarried out in a suitable solvent. The solvent can be an organic solventor mixture of one or more organic solvents, or a mixture of one or moreorganic solvents with water or an aqueous buffer. Examples of organicsolvents can be used include, but are not limited, to dimethylacetimide(DMA), methanol, ethanol, methylene chloride, acetone, DMF, DMSO, ethylacetate, dioxane, and acetonitrile.

In one embodiment, the reaction can be carried out in a mixture ofdimethylacetimide (DMA) and a succinate buffer. Preferably, thesuccinate buffer has a pH of about 5. More preferably, the reaction canbe carried out in a 60:40 (v:v) mixture of DMA and succinate bufferhaving a pH about 5.

The reaction can be carried out at a suitable temperature. In oneembodiment, the reaction is carried out at room temperature (about20-25° C.). Alternatively, the oxidation reaction can be carried out ata low temperature, for example, at about −20° C. to about 15° C. Inanother alternative, the reaction can be carried out at an elevatedtemperature, for example, higher than about 30° C.

The conjugates of the present invention can be prepared by reacting acell-binding agent with a compound of the present invention (e.g.,compounds of structural formula (I) and (II)) to provide a mixturecomprising a cell-binding agent-cytotoxic compound conjugate; theunreacted compound; and reaction side-products. The mixture isoptionally purified to provide a purified conjugate.

In another embodiment, the conjugates of the present invention can beprepared through a conjugate oxidation reaction by treating a conjugatesof structural formula (VII′), (VII) or (VIII) with an oxidant (seeScheme 3). The values and preferred values for variables depicted instructural formulas (VII′), (VII) and (VIII) are as described above forstructural formulas (III′), (III) and (IV), respectively.

The conjugate of structural formula (X) can be prepared by reactingconjugate comprising a thioether linking group represented by structuralformula (XI) with an oxidant.

For the conjugate oxidation described above, any suitable oxidantcapable of converting a thiol group to a sulfoxide group can be used.Examples of oxidant include, but are not limited to, NaIO₄, t-BuOCl,dioxiranes, calcium hypochlorite Ca(OCl)₂, sodium chlorite NaClO₂, HNO₃(optionally in the presence of a AuCl₄ ⁻ catalyst), O₂ (in the presenceof a ceric ammonium nitrate catalyst), acyl nitrites, sodium perborate,organic peroxides (e.g., ROOH, wherein R is a C₁₋₆ alkyl), and H₂O₂(optionally in the presence of a bis(acetylacetonato)oxovanadiumcatalyst). Preferably, the oxidant is H₂O₂ with abis(acetylacetonato)oxovanadium catalyst.

The conjugate oxidation reaction can be carried out at a suitabletemperature. In one embodiment, the reaction can be carried out at roomtemperature (about 20-25° C.). Alternatively, the oxidation reaction canbe carried out at a low temperature, for example, at about −20° C. toabout 15° C. In another alternative, the reaction can be carried out atan elevated temperature, for example, higher than about 30° C.Preferably, the oxidation reaction is carried out at about 4° C. Morepreferably, the oxidation reaction is carried out at about 4° C. forabout 1-12 hours.

In another embodiment, the mixture of the conjugate oxidation reactionis purified immediately after the reaction (e.g., for about 1-12 hours)to remove excess oxidant. Any suitable purification method can be used.In one embodiment, the purification method is as described herein. Inanother embodiment, the purification method is non-absorptivechromatography and/or dialysis. Preferably, the purification method isnon-absorptive chromatography with G25 desalting resin and/or dialysisat 4° C.

Any suitable purification methods can be used for purifying the mixturecomprising the conjugates of the present invention. In one embodiment,the mixture can be purified using tangential flow filtration (TFF),e.g., a membrane-based tangential flow filtration process,non-adsorptive chromatography, adsorptive chromatography, adsorptivefiltration, or selective precipitation, or any other suitablepurification process, as well as combinations thereof. One of ordinaryskill in the art will appreciate that purification after the conjugationstep enables the isolation of a stable conjugate comprising thecell-binding agent chemically coupled to the cytotoxic agent.

Any suitable TFF systems may be utilized for purification, including aPellicon type system (Millipore, Billerica, Mass.), a Sartocon Cassettesystem (Sartorius AG, Edgewood, N.Y.), and a Centrasette type system(Pall Corp., East Hills, N.Y.).

Any suitable adsorptive chromatography resin may be utilized forpurification. Preferred adsorptive chromatography resins includehydroxyapatite chromatography, hydrophobic charge inductionchromatography (HCIC), hydrophobic interaction chromatography (HIC), ionexchange chromatography, mixed mode ion exchange chromatography,immobilized metal affinity chromatography (IMAC), dye ligandchromatography, affinity chromatography, reversed phase chromatography,and combinations thereof. Examples of suitable hydroxyapatite resinsinclude ceramic hydroxyapatite (CHT Type I and Type II, Bio-RadLaboratories, Hercules, Calif.), HA Ultrogel hydroxyapatite (Pall Corp.,East Hills, N.Y.), and ceramic fluoroapatite (CFT Type I and Type II,Bio-Rad Laboratories, Hercules, Calif.). An example of a suitable HCICresin is MEP Hypercel resin (Pall Corp., East Hills, N.Y.). Examples ofsuitable HIC resins include Butyl-Sepharose, Hexyl-Sepaharose,Phenyl-Sepharose, and Octyl Sepharose resins (all from GE Healthcare,Piscataway, N.J.), as well as Macro-prep Methyl and Macro-Prep t-Butylresins (Biorad Laboratories, Hercules, Calif.). Examples of suitable ionexchange resins include SP-Sepharose, CM-Sepharose, and Q-Sepharoseresins (all from GE Healthcare, Piscataway, N.J.), and Unosphere S resin(Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable mixedmode ion exchangers include Bakerbond ABx resin (J T Baker, PhillipsburgN.J.). Examples of suitable IMAC resins include Chelating Sepharoseresin (GE Healthcare, Piscataway, N.J.) and Profinity IMAC resin(Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable dyeligand resins include Blue Sepharose resin (GE Healthcare, Piscataway,N.J.) and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, Calif.).Examples of suitable affinity resins include Protein A Sepharose resin(e.g., MabSelect, GE Healthcare, Piscataway, N.J.), where thecell-binding agent is an antibody, and lectin affinity resins, e.g.Lentil Lectin Sepharose resin (GE Healthcare, Piscataway, N.J.), wherethe cell-binding agent bears appropriate lectin binding sites.Alternatively an antibody specific to the cell-binding agent may beused. Such an antibody can be immobilized to, for instance, Sepharose 4Fast Flow resin (GE Healthcare, Piscataway, N.J.). Examples of suitablereversed phase resins include C4, C8, and C18 resins (Grace Vydac,Hesperia, Calif.).

Any suitable non-adsorptive chromatography resin may be utilized forpurification. Examples of suitable non-adsorptive chromatography resinsinclude, but are not limited to, SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™resins (e.g., S-200 and S-300), SUPERDEX™ resins (e.g., SUPERDEX™ 75 andSUPERDEX™ 200), BIO-GEL® resins (e.g., P-6, P-10, P-30, P-60, andP-100), and others known to those of ordinary skill in the art.

In Vitro Cytotoxicity

The cytotoxic compounds and cell-binding agent-cytotoxic agentconjugates of the invention can be evaluated for their ability tosuppress proliferation of various cancer cell lines in vitro. Forexample, cell lines such as the squamous cell carcinomal line A431, thelung carcinoma line PC9, the human colon carcinoma line COLO 205, therhabdomyosarcoma cell line RH-30, and the multiple myeloma cell lineMOLP-8 can be used for the assessment of cytotoxicity of these compoundsand conjugates. Cells to be evaluated can be exposed to the compounds orconjugates for 1-5 days and the surviving fractions of cells measured indirect assays by known methods. IC₅₀ values can then be calculated fromthe results of the assays. Examples of in vitro potency and targetspecificity of antibody-cytotoxic agent conjugates of the presentinvention are shown in FIGS. 1 and 2.

Compositions and Methods of Use

The present invention includes a composition (e.g., a pharmaceuticalcomposition) comprising novel cytotoxic compounds described herein(e.g., compounds of structural formula (I′), (I) and (II)), derivativesthereof, or conjugates thereof, (and/or solvates, hydrates and/or saltsthereof) and a carrier (a pharmaceutically acceptable carrier). Thepresent invention also includes a composition (e.g., a pharmaceuticalcomposition) comprising novel cytotoxic compounds described herein(e.g., compounds of structural formula (I′), (I) and (II)), derivativesthereof, or conjugates thereof, (and/or solvates, hydrates and/or saltsthereof) and a carrier (a pharmaceutically acceptable carrier), furthercomprising a second therapeutic agent. The present compositions areuseful for inhibiting abnormal cell growth or treating a proliferativedisorder in a mammal (e.g., human). The present compositions are alsouseful for treating depression, anxiety, stress, phobias, panic,dysphoria, psychiatric disorders, pain, and inflammatory diseases in amammal (e.g., human).

In one embodiment, the present invention includes a composition or apharmaceutical composition comprising the conjugates described herein(e.g., conjugates of structural formula (III′), (III) or (IV), whereinthe average molar ratio of the cytotoxic agent to the cell-binding agentof the conjugates in the composition is about 1 to about 10, about 2 toabout 7, about 3 to about 5, about 2.5 to about 4.5 (e.g., about 2.5,about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9,about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5), about3.0 to about 4.0, about 3.2 to about 4.2, about 4.5 to 5.5 (e.g., about4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1,about 5.2, about 5.3, about 5.4, about 5.5).

The present invention also includes a method of inhibiting abnormal cellgrowth or treating a proliferative disorder in a mammal (e.g., human)comprising administering to said mammal a therapeutically effectiveamount of cytotoxic compounds described herein (e.g., compounds ofstructural formula (I′), (I) and (II)), derivatives thereof, orconjugates thereof (e.g., conjugates of structural formula (III′), (III)or (IV)), (and/or solvates and salts thereof) or a composition thereof,alone or in combination with a second therapeutic agent.

The present invention also provides methods of treatment comprisingadministering to a subject in need of treatment a therapeuticallyeffective amount of any of the conjugates described above.

Similarly, the present invention provides a method for inducing celldeath in selected cell populations comprising contacting target cells ortissue containing target cells with an effective amount of acell-binding agent-cytotoxic compound conjugate of the presentinvention, a salt (e.g., pharmaceutically acceptable salt) or solvatethereof. The target cells are cells to which the cell-binding agent canbind.

If desired, other active agents, such as other anti-tumor agents, may beadministered along with the conjugate.

Suitable pharmaceutically acceptable carriers, diluents, and excipientsare well known and can be determined by those of ordinary skill in theart as the clinical situation warrants.

Examples of suitable carriers, diluents and/or excipients include: (1)Dulbecco's phosphate buffered saline, pH about 7.4, containing or notcontaining about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; and may also containan antioxidant such as tryptamine and a stabilizing agent such as Tween20.

The method for inducing cell death in selected cell populations can bepracticed in vitro, in vivo, or ex vivo.

Examples of in vitro uses include treatments of autologous bone marrowprior to their transplant into the same patient in order to killdiseased or malignant cells: treatments of bone marrow prior to theirtransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD); treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.

The conditions of non-clinical in vitro use are readily determined byone of ordinary skill in the art.

Examples of clinical ex vivo use are to remove tumor cells or lymphoidcells from bone marrow prior to autologous transplantation in cancertreatment or in treatment of autoimmune disease, or to remove T cellsand other lymphoid cells from autologous or allogenic bone marrow ortissue prior to transplant in order to prevent GVHD. Treatment can becarried out as follows. Bone marrow is harvested from the patient orother individual and then incubated in medium containing serum to whichis added the cytotoxic agent of the invention, concentrations range fromabout 10 μM to 1 pM, for about 30 minutes to about 48 hours at about 37°C. The exact conditions of concentration and time of incubation, i.e.,the dose, are readily determined by one of ordinary skill in the art.After incubation the bone marrow cells are washed with medium containingserum and returned to the patient intravenously according to knownmethods. In circumstances where the patient receives other treatmentsuch as a course of ablative chemotherapy or total-body irradiationbetween the time of harvest of the marrow and reinfusion of the treatedcells, the treated marrow cells are stored frozen in liquid nitrogenusing standard medical equipment.

For clinical in vivo use, the cytotoxic conjugate of the invention willbe supplied as a solution or a lyophilized powder that are tested forsterility and for endotoxin levels. Examples of suitable protocols ofconjugate administration are as follows. Conjugates are given weekly for4 weeks as an intravenous bolus each week. Bolus doses are given in 50to 1000 ml of normal saline to which 5 to 10 ml of human serum albumincan be added. Dosages will be 10 μg to 2000 mg per administration,intravenously (range of 100 ng to 20 mg/kg per day). After four weeks oftreatment, the patient can continue to receive treatment on a weeklybasis. Specific clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by one of ordinary skill in the art as the clinical situationwarrants.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of inducing cell death in selected cellpopulations include malignancy of any type including, for example,cancer of the lung (small cell and non-small cell), breast, colon,brain, prostate, kidney, pancreas, ovary, head and neck, skin(melanoma), Merkel cell carcinoma, glioblastoma, neuroblastoma, andcancers of lymphatic organs; autoimmune diseases, such as systemiclupus, rheumatoid arthritis, and multiple sclerosis; graft rejections,such as renal transplant rejection, liver transplant rejection, lungtransplant rejection, cardiac transplant rejection, and bone marrowtransplant rejection; graft versus host disease; viral infections, suchas CMV infection, HIV infection, AIDS, etc.; and parasite infections,such as giardiasis, amoebiasis, schistosomiasis, and others asdetermined by one of ordinary skill in the art.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (PDR). The PDR disclosesdosages of the agents that have been used in treatment of variouscancers. The dosing regimen and dosages of these aforementionedchemotherapeutic drugs that are therapeutically effective will depend onthe particular cancer being treated, the extent of the disease and otherfactors familiar to the physician of skill in the art and can bedetermined by the physician. The contents of the PDR are expresslyincorporated herein in its entirety by reference. One of skill in theart can review the PDR, using one or more of the following parameters,to determine dosing regimen and dosages of the chemotherapeutic agentsand conjugates that can be used in accordance with the teachings of thisinvention. These parameters include:

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Analogues and Derivatives

One skilled in the art of cytotoxic compounds will readily understandthat each of the cytotoxic compound described herein can be modified insuch a manner that the resulting compound still retains the specificityand/or activity of the starting compound. The skilled artisan will alsounderstand that many of these compounds can be used in place of thecytotoxic agents described herein. Thus, the cytotoxic agents of thepresent invention include analogues and derivatives of the compoundsdescribed herein.

All references cited herein and in the examples that follow areexpressly incorporated by reference in their entireties.

EXEMPLICIATION Example 1 General Methods for Preparing Conjugates of thePresent Invention Method 1:

A thiol-containing cytotoxic agent (e.g., D-SH) is reacted with amaleimide containing heterobifunctional linker (e.g., AMAS, BMPS, GMBS,EMCS, SBSV, PEG_(n)-Mal) in a mixture of dimethylacetimide (DMA) and 50mM succinate buffer pH 5 (60:40 DMA:buffer by volume) for 5 min togenerate a succinimidyl thioether. The mixture is then treated with mildoxidant (e.g., hydrogen peroxide, dimethyldioxirane) at 4° C., whichselectively converts thioether into sulfoxide and not the sulfone. Themixture is then added to an antibody solution buffered at pH about 6-9in a 3-7 fold molar excess (based on linker concentration) and thereaction was allowed to proceed overnight to generate the sulfoxidelinker conjugate.

Method 2:

An antibody-cytotoxic agent conjugate comprising a thioether group(preferably a succinimidyl thioether group) is treated at pH 7.4 with3-100 mole % of an oxidant (e.g., NaIO₄, t-BuOCl, dioxiranes, calciumhypochlorite Ca(OCl)₂, sodium chlorite NaClO₂, HNO3 and a AuCl₄ ⁻catalyst, O₂ and ceric ammonium nitrate catalyst, acyl nitrites, sodiumperborate, and organic peroxides ROOH and H₂O₂ with abis(acetylacetonato)oxovanadium catalyst). Conjugate oxidation is doneat 4° C. for 1-12 hours followed by immediate removal of excess oxidantusing G25 desalting resin and/or dialysis.

Conjugates made by method 1 or 2 can be assayed for extent of thioetheroxidation to sulfoxide by kinetic trapping of the released sulfenic acidintermediate. An exemplary procedure for kinetic trapping is describedbelow in Example 2.

The invention will now be illustrated by reference to non-limitingexamples. Unless otherwise stated, all percents, ratios, parts, etc. areby weight. Hydrogen peroxide (30% w/v in water), N-ethyl maleimide, and5,5-dimethyl-1,3,-cyclohexanedione (dimedone) were purchased from Sigma.Antibody drug conjugates (Ab-SMCC-DM1, Ab-PEG₄-mal-DM1, Ab-PEG₄-mal-DM4)were prepared according to published methods (Kovtun, et al, CancerResearch, 2010, 70, 2528-37). A Bruker ESQUIRE™ 3000 ion trap massspectrometer was used to obtain mass spectra for all reaction productsand was used in line with a Water 2695 series HPLC. Samples wereanalyzed using the analytical reverse phase HPLC method described below:

Column: Vydac C8 (208TP104), 250×4.6 mm, 5 micron. Temperature: 25° C.

Flow rate: 1.0 mL/min Injection volume: 50 microliters

Absorbance detection: 252 nm

Linear gradient of 80% A 80% B over 30 min

Solvent A: water (0.025% formic acid)

Solvent B: acetonitrile (0.025% formic acid)

Mass Spectrometer Conditions

Ionization method: Electrospray; Nebulizer gas flow: 25 psi;

Drying temperature: 350° C.; Heating gas flow: 8.0 L/min;

Mass range: 50-2000

MS detection in alternating positive and negative ion modes or in MS²mode.

Example 2 Selective Oxidation of Ab-SMCC-DM1 to Sulfoxide LinkedConjugate 2

chB38.1-SMCC-DM1 (9 μM in PBS, pH 7.4) was treated with 1 μM hydrogenperoxide and 5 mole % bis(acetylacetonato)oxovanadium catalyst at 4° C.for 4 h. To remove excess oxidant, gel fitration of the conjugate into aG25 Sephadex (GE healthcare) column equilibrated in 10 mM sodium citratepH 5.5 followed by dialysis (2 exchanges in a slide-a-lyzer cassette)was carried out. The oxidized conjugate was assayed for maytansine perantibody ratio (MAR), % monomer, and % unconjugated free maytansinoidand the sample was identical to the parent conjugate (3.7 MAR, 0.1% freemaytansinoid, 95% monomer).

In Vitro Cytotoxicity Assays Comparing Activity of Conjugate 1(Ab-SMCC-DM1) and Selectively Oxidized Conjugate 2

Confluent T75 flasks of PC9 cells (for anti-EPCAM-SMCC-DM1) and A431cells (for anti-EGFR-SMCC-DM1) grown in growth medium (DMEM (Invitrogen)containing 10% fetal bovine serum (ATCC), 2 mM L-glutamine (Invitrogen),and penicillin-streptomycin (Invitrogen) were detached with 0.25%tryspin-EDTA (Invitrogen), stained with trypan blue (Invitrogen) and thenumber of live cells was counted on a hemacytometer. Between 1000 and2000 cells/well were seeded in 96-well flat-bottom tissue culture plates(Falcon) in growth media and allowed to adhere overnight at 37° C. Allcells were obtained from ATCC. After 1 day, cells were continuouslyexposed to anti-EpCAM or anti-EGFR antibody conjugates (1 and 2) for 96hr at 37° C. at concentrations of 2 pM to 30 nM (3-fold serialdilutions). Blocking controls were performed in parallel where cellswere treated for 1 hr at 37° C. with 1 μM unconjugated antibody in freshgrowth media prior to conjugate exposure. Each test condition wasperformed in triplicate. After conjugate treatment, 20 μl WST-8 reagent(Dojindo Molecular) was added to each well and incubated at 37° C. for1-5 h for color development. The plate absorbance was measured at 450 nmand used to calculate the surviving cell fraction (compared withuntreated control cells). The IC₅₀ values were estimated from plots ofconjugate concentration (M) versus surviving cell fraction. Cytotoxicitydata shown in FIGS. 1 and 2. FIG. 1 shows that anti-EGFR antibodyconjugate IgG1-sulfoxide-DM1 conjugate 2 is ˜2-fold more potent than theparent IgG1-SMCC-DM1 conjugate 1. FIG. 2 shows thatanti-EpCAM-SMCC-sulfoxide-DM1 conjugate 2 is ˜3-fold more potent thanthe parent anti-EpCAM-SMCC-DM1 conjugate 1. Preincubation of PC9 cellswith 1 μM unconjugated anti-EpCAM antibody (to fully block antigenbinding sites) leads to significant reduction in cell killing activityfor conjugates 1 and 2. Therefore, the data suggest that the additionalactivity observed for conjugate 2 is antigen-dependent.

Characterization of Oxidized Thioether in Conjugate 2

Conjugates made by method 1 or 2 were assayed for extent of thioetheroxidation to sulfoxide by kinetic trapping of the released sulfenic acidintermediate. Sulfoxide linker conjugate 2 (36 μM) was incubated with 10mM dimedone (12 h, 37° C., PBS, pH 7.4). Free maytansinoid species werethen acetone extracted and identified by LC-MS and quantified (byintegrated area at 252 nm and comparison to a standard curve). Presenceof maytansinoid-dimedone adduct (i.e. DM1-dimedone) was evidence ofoxidation of the thioether ether linkage selectively to the sulfoxide.Data shown in FIG. 4.

Example 3 Oxidation Promoted Release of Maytansinoids from ThioetherLinker Ab-PEG₄-mal-DM_(x)

Ab-PEG₄-mal-DM1 and Ab-PEG₄-mal-DM4 (36 μM, average of 4 maytansinoidsper antibody) with no detectable unconjugated maytansinoid was treatedwith hydrogen peroxide (0.1 μM-50 μM final concentration) at pH 7.4(PBS) for 12 h at 37° C. Increase in free maytansinoids was quantifiedusing a mixed-mode chromatography method (Fleming, et al, AnalyticalBiochemistry, 2005, 340, 272). Briefly, a HISEP shielded hydrophobicphase column (5 μm particle size, 4.6×250 mm length, Supelco,Bellefonte, Pa., USA) was used for analyzing Ab-DM1 conjugates.Detection was at 252 and 280 nm (extracted from PDA spectra). The flowrate was 0.7 ml/min. Mobile phase A consisted of 100 mM ammonium acetate(pH 7.0). Mobile phase B was 100% acetonitrile. The column wasequilibrated at 25% B followed by a linear gradient over 25 min to 40% Bafter sample injection. Intact conjugate elutes between 2-5 min whilereleased maytansinoid is detected between 10-25 min. Data shown in FIG.5C.

Example 4 Rate of Oxidation of DM1-NEM and DM4-NEM

10 μM DM1-NEM or DM4-NEM (95% PBS pH 7.4, 5% dimethylacetamide) weretreated at room temperature with 1 mM hydrogen peroxide and reactionaliquots (30 μl) were injected directly onto RP-HPLC at various timepoints from 0-400 min. Maytansinoid products were quantified bymeasuring integrated area under the curve at 252 nm and comparison to acalibration curve with known standards. Kinetic data shown in FIG. 5A.

Example 5 Rate of Sulfoxide Elimination of DM1-SO-NEM and DM4-SO-NEM

8 μM DM1-SO-NEM or DM4-SO-NEM (95% PBS pH 7.4, 5% dimethylacetamide)were incubated at 37° C. and reaction aliquots (50 μl) were injecteddirectly onto RP-HPLC at various time points from 0-12 h. Maytansinoidproducts were quantified by measuring integrated area under the curve at252 nm and comparison to a calibration curve with known standards.Kinetic data shown in FIG. 5B.

Thioether formed with hindered thiol DM4 oxidizes 2-fold slower (FIG.5A) and undergoes sulfoxide elimination 2-fold slower than correspondingthioether formed from unhindered DM1 thiol (FIG. 5B). The observationthat Ab-PEG4-mal-DM4 is ˜5-fold more resistant to oxidation promotedfree maytansinoid release than Ab-PEG4-mal-DM1 (FIG. 5C) is consistentwith the observed rates of sulfoxide formation and beta-elimination.

1. A compound represented by the following structural formula:

wherein: D is a cytotoxic agent; X is S or Se; R and R′ are eachindependently selected from the group consisting of —H, an alkyl, acycloalkyl, —OR^(a), and —NR^(b)R^(c); R^(a), R^(b) and R^(c) are eachindependently H or an alkyl; p is 0, 1 or 2 Y is an alkylene, apolyethylene glycol unit represented by —(CH₂—CH₂—O)_(n)—R^(d)—,—R^(d)-E- or —R^(d)—W—R^(e)—, n is an integer from 1 to 24; E is acycloalkyl, a heterocyclyl, an aryl or a heteroaryl, W is —C(═O)NH—,—NHC(═O)—, —(C═O)O— or —O(C═O)—, R^(d) is absent or an alkyl; R^(e) isan alkyl; M is a linking group that can react with a cell-binding agentto form a covalent bond.
 2. The compound of claim 1, wherein D is amaytansinoid.
 3. The compound of claim 2, wherein D is represented bythe following structural formula:

wherein: Y₁ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂—, R₁, R₂,R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, an alkyl, analkenyl, a cycloalkyl, a heteroaryl, a heterocyclyl, or an aryl; and l,m and n are each independently 0 or an integer from 1 to
 5. 4. Thecompound of claim 3, wherein R₁ and R₂ are each independently H, C₁₋₄alkyl or C₁₋₄ alkyl substituted with one to six halogens.
 5. Thecompound of claim 4, wherein R₁ and R₂ are each independently H, methyl,—CF₃ or —CCl₃.
 6. The compound of claim 4, wherein R₁ and R₂ are both Hor methyl.
 7. The compound of claim 3, wherein l and m are 0, n is 1,R₁, R₂, R₃ and R₄ are all H.
 8. The compound of claim 3, wherein n is 0,l and m are both 1, R₅, R₆, R₇ and R₈ are all H, R₁ and R₂ are bothmethyl.
 9. The compound of claim 3, wherein n is 0, l and m are both 1,R₁, R₅, R₆, R₇ and R₈ are all H, and R₂ is methyl.
 10. The compound ofclaim 1, wherein X is S.
 11. The compound of claim 1, wherein p is 0.12. The compound of claim 1, wherein one of R and R′ is H, and the otheris H, a C₁₋₄ alkyl, —OR^(a), or —NR^(b)R^(c), wherein R^(a), R^(b) andR^(c) are each independently H or a C₁₋₄ alkyl.
 13. The compound ofclaim 12, wherein R and R′ are both H.
 14. The compound of claim 1,wherein Y is a C₁₋₆ alkylene, a polyethylene glycol unit represented by—(CH₂—CH₂—O)_(n)—R^(d)—, —R^(d)-E- or —R^(d)—W—R^(e)—, wherein R^(d) isabsent or a C₁₋₄ alkyl; R^(e) is a C₁₋₄ alkyl; W is —C(═O)NH—; E iscyclohexyl and n is an integer from 2 to
 8. 15. The compound of claim14, wherein Y is


16. The compound of claim 1, wherein M represents a maleimide, ahaloacetamido, —SH, —SSR^(f), —CH₂SH, —CH(Me)SH, —C(Me)₂SH, —NHR^(g),—CH₂NHR^(g), —NR^(g)NH₂, —COOH, a reactive ester, an amino acid or apeptide comprising 2 to 10 amino acids, wherein R^(f) is selected fromphenyl, nitrophenyl, dinitrophenyl, carboxynitrophenyl, pyridyl ornitropyridyl and R^(g) is —H or a C₁₋₄ alkyl.
 17. The compound of claim16, M represents a maleimide, haloacetamido, N-hydroxysuccinimde ester,N-hydroxy sulfosuccinimide ester, nitrophenyl ester, dinitrophenylester, sulfo-tetraflurophenyl ester or pentafluorophenyl ester.
 18. Thecompound of claim 17, wherein M represents a N-hydroxysuccinimide ester.19-83. (canceled)